Wani K A et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Genetic analysis of dopamine signaling in C. elegans"

Chase D L, Wani K A

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Dopamine acts through G-protein-coupled D1-and D2-like receptors to modulate neuronal activity. Defects in dopamine signaling underlie neurological disorders including Parkinson's disease, schizophrenia and drug addiction. Despite its clinical relevance, detailed understanding of how dopamine exerts its functions remains largely unknown. To identify the molecular mechanisms of dopamine signaling, we have taken a genetic approach using C. elegans as a model system. In C. elegans, dopamine acts through the D2-like receptor, DOP-3, and the G-alpha inhibitory G-protein, GOA-1, to inhibit locomotion in response to environmental cues. This inhibition of locomotion is also observed when animals are exposed to exogenous dopamine with high concentrations causing paralysis. The paralytic effect of exogenous dopamine acts through the DOP-3 receptor and allows us to perform robust genetic screens to identify novel components of dopamine signaling. Null mutants lacking DOP-3 receptor are partially resistant to paralysis, while null mutations in GOA-1, which is coupled to DOP-3, cause complete resistance to exogenous dopamine. This suggests that there is another dopamine receptor in C. elegans also coupled to GOA-1 that acts to inhibit locomotion. In order to identify this unknown receptor and its downstream effectors, we conducted an enhancer screen in which we mutagenized dop-3 receptor mutants to identify second-site mutations that cause complete resistance to the paralytic effects of exogenous dopamine. We screened 92,000 mutagenized haploid genomes and isolated five mutants. All five mutants are more resistant to exogenous dopamine than dop-3 single mutants and their resistance is similar to goa-1 mutants. Mapping of these mutations using single-nucleotide polymorphisms followed by complementation analysis resulted in the identification of an already-known dopamine signaling component as well as four novel genes. We are currently fine-mapping these four mutations to a region of a chromosome.

Chun-Yi Huang et al. (2007) International Worm Meeting "eif-3.K is a pro-apoptotic gene in C. elegans."

Jia-Yun Chen, Yi-Chun Wu, Chun-Yi Huang

[International Worm Meeting, 2007]

Programmed cell death (or apoptosis) is an important feature of C. elegans development. Previous studies have identified pro-apoptotic genes egl-1, ced-3 and ced-4 and anti-apoptotic genes ced-9 and icd-1 that control programmed cell death.. We have identified and characterized a novel pro-apoptotic gene eif-3.K. Loss-of-function by mutation or RNAi inactivation in eif-3.K resulted in a decrease of cell corpses, whereas heatshock-induced over-expression of eif-3.K weakly but significantly increased cell corpses. Interestingly, the eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 or ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the programmed cell death pathway. Using a cell-specific promoter to express eif-3.k in touch neurons, we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. To further explore EIF-3.K function, we generated antibodies against bacterially expressed EIF-3.K protein. We found that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. As human eif-3.K can functionally substitute C. elegans eif-3.K in an eif-3.K mutant, the function of eif-3.K in apoptosis is likely conserved in evolution.

Chun-Yi Huang et al. (2006) East Asia C. elegans Meeting "eif-3.K is a pro-apoptotic gene in C. elegans"

Teng-Wei Huang, Chun-Yi Huang

[East Asia C. elegans Meeting, 2006]

Programmed cell death or apoptosis is an important feature during C. elegans development. The pro-apoptotic genes egl-1, ced-4 and ced-3 are required for the execution of cell death. We have identified and characterized a novel pro-apoptotic gene eif-3.K. A loss-of-function mutation or inactivation by RNA interference in eif-3.K resulted in a reduction of cell corpse number during embryogenesis, whereas heatshock-induced over-expression of eif-3.K weakly but significantly promoted programmed cell death. In addition, eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 and ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the genetic pathway during programmed cell death. Using a cell-specific promoter we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. We generated antibodies against bacterially expressed EIF-3.K protein. The immunostaining result showed that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. To better understand the cell-death defect of eif-3.K mutants, we are currently performing a 4D microscopic analysis of the cell death process in wild-type and eif-3.K mutants.

Johnson, Christina K. et al. (2019) International Worm Meeting "Requirement of glial Na+/K+-ATPase in touch sensation highlights ionic and metabolic link between glia and touch neurons in C. elegans."

Johnson, Christina K., Bianchi, Laura, Wang, Ying, Fernandez Abascal, Jesus

[International Worm Meeting, 2019]

Isolated microenvironments, such as the tripartite synapse, where the concentration of ions is regulated independently from the surrounding tissues, exist throughout the nervous system, including in mechanoreceptors. Modulation of the ionic composition of these microenvironments has been suggested to be achieved by glia and other accessory cells. However, the molecular mechanisms of ionic regulation and effects on neuronal output and animal behavior are poorly understood. Using the model organism C. elegans, our lab published that Na+ channels of the DEG/ENaC family expressed in glia control neuronal Ca2+ transients and animal behavior in response to sensory stimuli. DEG/ENaC Na+ channels are known to establish a favorable driving force for K+ excretion, which occurs via inward rectifier K+ channels, in epithelial tissues across species. We hypothesized that a similar mechanism exists in the nervous system. Using molecular, genetic, in vivo imaging, and behavioral approaches, we showed that expression in glia of inward rectifier K+ channels and cationic channels rescues the sensory deficits caused by knock-out of glial DEG/ENaCs without disrupting neuronal morphology, supporting our hypothesis. Based on this model, Na+/K+-ATPases are also needed to maintain ionic concentrations following influx of Na+ and excretion of K+. We show here that, in addition to glial Na+ and K+ channels, two specific glial Na+/K+-ATPases, EAT-6 and CATP-1, are needed for touch sensation and that their requirement can be bypassed by a high glucose diet. The effect of glucose is dependent on ATP binding capability of the pump, translation, transcription, and the activity of CATP-2, a third Na+/K+-ATPase ?-subunit. Taken together, our results support metabolic and ionic cooperation between glia and neurons in C. elegans mechanosensors, a mechanism that is essential to regulating neuronal output and may be conserved across species.

J Ceron et al. (2004) European Worm Meeting "IDENTIFICATION OF GENES ACTING REDUNDANTLY WITH LIN-35 RB"

S van den Heuvel, J Rual, K Wani, M Vidal, J Ceron, A Chandra

[European Worm Meeting, 2004]

lin-35 encodes the single C. elegans representative of the Retinoblastoma (Rb) protein family, which consist of three members in mammals (pRb, p107, p130). Rb has been shown to act in the transcriptional repression of genes with roles in cell cycle control, differentiation and apoptosis. Importantly, components of the "Rb pathway" are mutated or deregulated in most human cancers. Although lin-35 loss of function results in viable animals that predominantly show a reduced brood size, there are additional defects that become apparent when other genes are inactivated. For instance, a multivulva phenotype is observed when lin-35 loss of function is combined with inactivation of genes of a second class. Therefore, lin-35 functions as a SynMuv (synthetic multivulva) class B gene to repress vulval cell fates redundantly with SynMuv class A genes. More recently, redundant functions of lin-35 in cell-cycle control and pharyngeal morphogenesis have also been discovered (Boxem M and van den Heuvel S, 2001; Fay DS et al, 2003). In order to find additional genes that genetically interact with lin-35, we are carrying out novel forward and reverse genetic screens for phenotypes that are synthetic, suppressed or enhanced in the context of lin-35 loss of function. In these screens, we may identify genes that are required for cell survival in the absence of lin-35 function, revealing potential targets for anti-cancer therapeutics. In addition, genes may be found that act to regulate lin-35 function, or that act in parallel to lin-35. In the forward genetic screen of ~2850 haploid genomes, we isolated 20 mutants that showed interaction with lin-35 Rb. These included 6 SynMuv class A mutants, which validates the screen. For the reverse genetic approach, we have screened more than 10,000 RNAi feeding clones of the ORFeome RNAi library. This RNAi screen has identified a large number of genes, most of which display a feeding RNAi phenotype in lin-35 mutants but not in wild-type animals. We are using different genetic backgrounds and other gene inactivation methods in secondary screens to further select true candidates. Encouragingly, cki-1 (known to interact genetically with lin-35) has already been identified in the reverse screen.

Maelle Jospin et al. (2001) International C. elegans Meeting "Basic electrophysiological properties of body wall muscle from the Nematode C elegans."

Maelle Jospin, Laurent Segalat, Bruno Allard

[International C. elegans Meeting, 2001]

Electrophysiological properties of striated muscle cells were investigated with the patch clamp technique in the Nematode C elegans . Worms were immobilised with cyanoacrylate glue and longitudinally incised using a tungsten rod sharpened by electrolysis. Recording pipettes were sealed on GFP-expressing body wall muscle cells. In the whole cell configuration, under current clamp conditions, in the presence of Ascaris medium in the bath and K-rich solution in the pipette, no action potential could be induced in response to current injection. Under voltage clamp control and in the same ionic conditions, depolarizations above -30 mV from a holding potential of -70 mV gave rise to outward K currents. Outward K currents resulted from two components, one fast inactivating component blocked by 4-aminopyridine, one delayed sustained component blocked by tetraethylammonium. In the presence of both blockers, an inward Ca current was revealed and inhibited by cadmium. Single channel recording using the inside-out configuration revealed the existence of a Ca-activated Cl channel and a Ca-activated K channel. Single channel experiments are currently performed to characterise voltage-gated conductances at the unitary level.

K Omata et al. (1999) International C. elegans Meeting "Habituation of C. elegans for the touch sensitivity"

K Kawamura, F Yonezawa, K Omata

[International C. elegans Meeting, 1999]

In order to study the habituation of C. elegans for the touch sensitivity, we carry out computer simulations, in which the neural circuit is formed by making use of the data table constructed recently by Oshio et al [1]. The i -th neuron is connected with the neighboring j -th neuron through the coupling strength K ij , which is varied dynamically by the Hebb rule. Note that K ij is not necessarily equal to K ji because there are one-way connections between the neurons by chemical synapses. As a reference state, we first deal with the neural circuit consisting only of the neurons ALM, AVM, PLM, PVM, AVA, AVB, PVC, AVD, A and B, that are related to the forward and backward movement directly. We give periodic stimuli to the sensory neurons PLM, PVM, and monitor the response of the motor neuron A. We find that the frequency of the response decreases with time, which indicates that the habituation to the touch sensitivity actually takes place. As one deviation from the reference state, we kill the inter-neuron AVD, and perform the same analysis described in the above. There is a tendency that the decay of the response curve becomes faster, and the habituation is enhanced. As the other deviations, there are several possibilities of killing the inter-neurons AVA, AVB, PVC and/or AVD. We discuss the enhancement of the habituation in relation with the recent experimental results by Hosono. [1.] K. Oshio, S. Morita, Y. Osana and K. Oka; C. elegans synaptic connectivity data'', Technical Report, CCEP, Keio Future No.1 (1998)

Jamie White et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Neurons necessary for sex-specific social behaviors"

Thomas Nicholas, Eliott Davidson, Jamie White, Erik Jorgensen

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

C. elegans nematodes sense and respond to other individuals in their environment via signals released by hermaphrodites. The response is sex-specific: males are attracted to the signals and hermaphrodites avoid them. This is interesting because the male and hermaphrodite nervous systems have highly similar anatomy, and the stimulus detected by each is the same--hermaphrodite-derived signals--but the response is opposite. This could arise from (1) sex-specific neurons, (2) sex-specific connectivity of anatomically similar neurons, (3) sex-specific physiology of similar neurons, or (4) a combination of these mechanisms. We are therefore interested in which neurons are necessary and sufficient for male-attraction and hermaphrodite avoidance behaviors. To determine this, we are using a combination of mutant analysis, neuron-specific rescue, and laser ablation. Avoidance requires tax-4 and osm-3. These genes overlap in the ASE, ASG, ASI, ASJ, and ASK pairs. Avoidance behavior is normal in unc-130 (required for the ASG fate) and in che-1 (required for proper ASE fate) mutants, indicating that ASI/J/K are most likely necessary for avoidance behavior. Avoidance can be rescued in a tax-4(p678) background by tax-4(+) expression from an odr-4 promoter, which includes expression in ASG and ASI/J/K but not ASE, indicating that ASI/J/K are sufficient for avoidance. ASI/J/K will be laser-ablated in the rescued strain to show which are necessary. Attraction behavior requires osm-9. Attraction behavior can be partially rescued in an osm-9(ky10) background by osm-9(+) expression in either male-specific neurons (pkd-2 promoter), or by expression in the AWB, AWC, and ASI/J/K pairs (odr-1 or daf-11 promoters). This indicates these two sets of neurons are each partially sufficient for attraction behavior. Of the second set, attraction is normal in ceh-36 mutants (required for AWC fate), indicating the AWB and/or ASI/J/K pairs are sufficient for partial rescue. We predict full rescue will be obtained by a combination of expression from the pkd-2 and odr-1/daf-11 promoters. Laser ablation will be carried out in the fully rescued strain to show which neurons of the sufficient set are necessary. It appears, then, that the different sexes may use the same neurons--one or more of the ASI/J/K pair--to generate sex-specific behaviors, but males need at least some input from sex-specific neurons.

Motomichi Doi et al. (2005) International Worm Meeting "Regulation of Acetylcholine receptor clustering at excitatory synapses by Na+/K+ ATPase"

Kouichi Iwasaki, Motomichi Doi

[International Worm Meeting, 2005]

Proper localization of neurotransmitter receptors at synapses is crucial for establishment of neural circuits in both central and peripheral nervous systems. The C. elegans body-wall muscles express two acetylcholine (Ach) receptors and one GABA receptor, which should be localized to specific postsynaptic sites corresponding to presynaptic neuron types. However, precise molecular mechanisms of how these receptors are localized at appropriate postsynaptic sites have not been understood well. Here we show that the Na+/K+ ATPase is required for Ach receptor localization at excitatory synapses. The Na+/K+ ATPase belongs to the P-type ATPase group, which catalyzes active transport of cations across the plasma membrane by using energy of ATP hydrolysis, and is implicated in neurodegeneration and several human neurological disorders such as familial hemiplegic migraine. In the course of studying the function of the Na+/K+ ATPase, we have found that the eat-6 (Na+/K+ ATPase subunit) and nkb-1 (1 subunit) mutants are hypersensitive to the drugs that affect cholinergic synaptic transmission. We treated these mutants with aldicarb, an Ach esterase inhibitor, and levamisole, an Ach agonist. Both drugs paralyzed the mutants significantly faster than wild-type animals, suggesting that these mutants have abnormally high Ach sensitivity at the postsynaptic body-wall muscles. Therefore, we have hypothesized that these mutations cause a higher expression of the Ach receptors at the postsynapse compared with the wild type. To test this hypothesis, we examined localization of UNC-29::GFP in the body-wall muscle; unc-29 encodes a non- nicotinic Ach receptor subunit. In the wild type, strong GFP signals accumulated inside muscle cell bodies with occasional puncta along the ventral nerve cord. In the eat-6 and nkb-1 mutants, however, strong GFP puncta are observed along the ventral nerve cord with little accumulation inside the muscle, suggesting that the Na+/K+ ATPase is required for clustering the Ach receptor at a postsynapse. This effect is specific to the Ach receptor since the GABA receptor (UNC-49B::GFP) localization in these mutants was not affected. Furthermore, we found no significant differences in overall presynaptic architectures between the wild type and these mutants by examining the expression of SNB-1::GFP. Our findings strongly suggest that the Na+/K+ ATPase specifically regulates either clustering or expression/internalization of the Ach receptor at excitatory synapses and might provide a clue for neurological disorders caused by the Na+/K+ ATPase mutations in human.

RIVA, C. et al. (2019) International Worm Meeting "A natural transdifferentiation event involving mitosis is empowered by integrating signaling inputs with conserved reprogramming factors."

Gally, C., Jarriault, S., Hajduskova, M., RIVA, C.

[International Worm Meeting, 2019]

Transdifferentiation is the direct conversion of one differentiated cell type into another, with or without cell division. In C. elegans the Y rectal cell naturally transdifferentiates into the PDA motor neuron with high efficiency, robustness and irreversibly and this process requires evolutionary conserved reprogramming factors. In this study our goal was to define in C. elegans which core and event-specific factors are affecting different natural transdifferentiations (Td). Looking at the worm's somatic cell lineage (Sulston et al., 1977), we postulated that the formation of the DVB GABAergic neuron from the K.p cell after K rectal cell division is another putative Td event. We confirmed that it is bona fide Td by analysing the cellular morphology and identity-specific markers in K and DVB. We assessed the role of Y-to-PDA reprogramming factors in K-to-DVB through genetic analyses. Based on unc-47 expression and presence of an axon, we found that sem-4, egl-5, sox-2 and ceh-6 are also involved in K-to-DVB, suggesting that these factors have conserved reprogramming properties in the worm. The characterization of sem-4 mutant revealed a defect in the erasure of the K.p initial rectal identity and in the gain of the neuronal one. We have found that during K-to-DVB a stereotyped, asymmetric cell division (ACD) occurs, with the K.p cell budding off. We have therefore investigated the potential role of cell division in this reprogramming event. The asymmetry of K division led us to assess the role of Wnt pathway in K-to-DVB. By testing lin-17, beta-catenins and pop-1 mutants, we found that indeed the Wnt pathway is involved. In lin-17(Ø), ACD is prevented and K.p displays an epithelial phenotype with big nucleus, expression of epithelial markers and absence of neuronal markers. In addition, we found that an important role of Wnt activity is to turn on expression of the lim-6 terminal selector in K.p. Finally, we found that Wnt and the reprogramming factor sem-4 act through two different pathways. These results demonstrate that during natural transdifferentiation the presence of an asymmetric cell division is not enough to allow cell identity change, but reprogramming factors are required in parallel to confer cell plasticity.